Purification of luciferase by affinity elution chromatography on Blue Dextran columns

Jomxal qf Chromarogrupin_.143 (1951) Else\& Scicaritic PubIishing Company,

16-I-167

Amsterdam

-

Printed

in The Netherlands

CZ-;RO>I. I-?X7

Note

Purification BIue Dextran Comparison

of luciferase columns of Sepharose

by

affinity

and silica

elution

as support

chromatography

on

matrices

The popularity and significance of the luciferase-catalysed bioluminescence in the fir&y is weI1 documented_ Its remarkable specificity and sensitivity towards adenosinr triphosphate (XTP) has induced us to \vork out methods for the purification of luciferase. The essence of the reaction is the production of light from luciferin in the presence of ATP. ivlg’ - and molecular oxygen as shown in eqns. 1 and 2 LHI

+ X-I-P + E +

E-LH2-AMP

E-LH2-AMP

+ O2 --, E f

f

PP

(I)

P + CO2

(2)

\\here LH, = luciferin. E = enzyme, E - LH, - XlMP = the luciferyl adenylate complex. P = the product osyluciferin and PP = the pyrophosphate. Several partial and complete luciferase purifications have been reported’-‘. Ho\ve\er_ some of them require len$hchromatographic procedures while others do not yield complete separation. This communication describes an improved separation technique for luciferaseRecent literature on nucIeotide-dependent enzymes deals with their purification by afiinity elution chromatography on Blue Destran Sepharose. a semi-specific high-moIecuIar-weight compound substituted with the mono-chiorotriazinyl dye Cibachron Blue F3GA which can mimic a polynucleotide, presumably due to an attracrion of the blue chromophore to a nutleotide-bindins site of the enzyme_ It has been. reported by Thompson et u[_~ that Blue Destran linked to Sepharose may be used as an affinity chromatographic medium for many proteins which bind dinucleotides and XTP. ESPERIXIESTXL

Cyanogen bromide-activated Sepharose 4B was purchased from Pharmacia. firefly lantern, Blue Destran and ATP from Sigma and DTT from E. Merck. All other chemicals u-ere of reagent grade. Sut22ple

preparf7tion

Extraction cw1-96:3;s1

of firefly

~oooo-oooo;So2.75

abdomen c

was carried

out accordins

19S? Elsesder Scientific Publishing

Company

to the method

of

KOTES

165

Antonik’ with slight modifications. A I-ml volume of dithiothreitol (DTT) (I95 mg/ml) was added per 100 mg of firefly abdomen at the end of the centrifugation. This was necessary for the stability of the crude extract. The preparation was dialysed against 0.02 1U Tris-acetate, pH 7.5 with 1 mlU EDTA for 24 h with three changes of the buffer_

Lrrcijiwa-e assay Lucifrrase activity was estimated by PICO ATP apparatus (Jobin Yvon, France)_ The ieaction mixture containing 50 ~1 of 10’ pg/ml ATP in 0.01 Al iMOPS (3-N-morpholinopropanesulphonic acid), pH 7.4 containing 10 rn?U MgSO,, 50 ~1 of luciferin (0.165 pg/ml) and 50 ~1 of luciferase sample was introduced in a special cuvette in the PICO ATP apparatus at 1S-20°C and the intensity of light in millivolts during the first 2 set of emission was recorded by a photomultiplier. The unit of activity is the maximum intensity of light recorded in millivolts pzr picogram ATP per milligram protein at 562 nm.

Blue Destran w;fs coupled to osiran silica by the procedure of Chang et a1.6. Blue Dextran Sepharose was prepared by the method of Ryan and Vestling’. The efficiency of the coupling reaction was controlled by measuring the differential absorbance in the visible region of the coupled gel suspended in 87 “/, glycerine_ A 0%ml aliquot of the dialysed extract was injected into the column of Blue Destran coupled to Sepharose or silica and the elution was started with O-01 &i MOPS containing 20 rn&~ MgSO, and 1 m&i DTT and continued until no protein was detected in the fractions. Then the eluent was changed to 0.5 miU ATP in 0.01 11~MOPS containing 20 rn&~ MgSOa and 1 mtU DTT to desorb the enzyme. The ballast proteins were stripped by eluting with a 3 &Z KC1 solution_ After each use, the columns were washed with four volumes of 3 iII KC1 and then exhaustively equilibrated with the starting eluent, 0.01 &f IMOPS, pH 7.4 containing 20 mlU lMgSOa and 1 mlU DTT, before the start of the next cycle_ Linder these conditions no loss of the blue chromophore was observed_ The same resin can be successfully used for more than six elutions. Dia&.sis of the fracrioas The fractions (1.0 ml) were pooled

into groups of three and dialysed against 0.02 IU Tris-acetate, pH 7.8 with 1 m&f EDT-4 for 16-19 h. All operations were carried out at 4;C. Each pooled fraction was tested for luciferase activity and protein concentration_ The absorbance at 275 nm was corrected against ATP in IMOPS. Sodium dodecyi suiplzate (SDS)-gel

electrophoresis and isoelectric focrtsiug

The homogeneity of the preparation of purified luciferase was checked by SDSgel electrophoresis in 7-5 “/gpolyacrylamide gel, pH S-O-9-O. Isoelectric focusing was carried out in the range pH 3.5-10.0 with 8 iU urea on 360 pm thick polyacrylamide gels freshly prepared on a cellophane support according to Gorg et a/.*. RESULTS

A comparison of chromatography on Blue Destran coupled to Sepharose and silica is shown in Table I. The elution profiles are shown in Figs. 1 and 2.

166

NOTES

Fis. 1. Elution profile of luciferase on Blue Dextran coupkd to Sepharose. A column of Blue Destran Sepkarose cquiIibrrtted with 0.01 _%IMOPS containing 20 m.tl MgSO, and 1 m.\l DTT, pH 7.4 was loaded with 0.3 ml ofcrude Iuciferase. The elucnt was changed to 0.01 .!I MOPS containing 20 mM MgSOG, 1 m.lf DTT and 0.5 mM ATP. Column frzstions ( 1 ml) wxe zssayed for enzyme ncti\ity and protein concentranon (see Experimental). -. Absorbrince st 775 nm: ---_ luciferase activity_

It is evident that Blue Destran coupled to sihca is more favourable for luciferase purilication since we Were able to recover about 300”; of the initial activity compared to about IS0 0, in the case of Sepharose Blue Destran. The purification in the two cases is 61-fold and Sl-fold respectively for silica- and Sepharose-based derivatives (Table I)_ This method is superior to that of Lundin er aZ_‘. who used (NH,),SO, precipitation followed by isoelectric focusing but obtained only a S. l-fold purified enzyme with 65”, of the initial activity_ We found good evidence for the homogzneity of our purified sample on Blue Destran Sepharose and silica by SDS-gel electrophoresis. Isoelectric focusing showed the presence of one band at a pH of about 5-O_ The purified fractions did not show the presence of other contaminating enzymes present in the extract. DISCUSSIOS

Blue Destran is known to complex with a wide range of proteins because it is specific for a super-secondary structure called the dinucleotide fold. This structure forms the ATP-binding site in phosphoplycerate kinase” and SAD-binding sites in lactate dehydrogenase and many other enzymes’ ‘. In our studies. the nucleotide ATP which has the highest affinity for luciferase

Fig. 2. Chromato_mm Dextran Sepharose.

of luciferase

ptied

on Blue Dextran

siicz

Conditions

and curves as for Blue

NOTES TABLE

167

I

PURIFICATION Pur$carion

OF LUCIFERASE

swps

ON BLUE DEXTRAN

Total prorein

COUPLED

TO SEPHAROSE

AND

SptTific

TornI acrid-v

acthitj

-4bsoiute

5; Initial

d bsolute value

% Initial

100

243,000

vaIue (nrg) 1 Crude extract 2 Dialysed extwct 3 Purified sample from Blue Deatran sepharose 1 Purified fraction from Blue Deatran silica

SILICA Purifirarion factor

1.5

5.S

247,300

100 102

9346 164,900

0 17.0

0.96

3.7

460.100

150

479.300

51.0

1.3

5.2

742,500

300

571,400

61.0

25.6

most effective in eluting the bound enzyme_ The optimum concentration of ATP was 0.5 mM_ By contrast, NaCl at a concentration at least 200 times that of the specific ligand ATP was required to desorb the enzyme from the column. Thus l&and specificity for luciferase was made use of to distinguish binding to the dinucleotide fold from non-specific ionic bindin,.= In our case, the relative affinity of Blue Destran and ATP for the same active site of luciferase must have facilitated the selective elution of the enzyme from the presumably large number of proteins and other compounds in the firefly abdomen homogenate which are bound to the affinity column by non-specific interactions. In a previous comparison of metal-chelate adsorbents based on Sepharose and silica. for the separation of nucleotides, a higher capacity was reported for silicabased adsorbent”. We find the same behaviour in this case, as the Blue Destran silica column has a higher enzyme-binding capacity than the corresponding Sepharosebased adsorbent_ The affinity status of these Blue Destran columns remains controversial iMany authors have recently pointed out that these are in fact pseudoaffinity interactions_ However it is too premature to draw any conclusion on the details of the interaction or to state the mode of inhibition of luciferase with respect to Blue Destran and ATP_ in the presence of ATP, the equilibrium between free enzyme and enzyme bound to Blue Destran could be shifted towards free enzyme. We confidently predict the evidence of a dinucleotide fold in firefly luciferasewas

REFERENCES 1 2 .< 1 5 6 7 S 9

M. Benny and M. Dolivo. FEBS Lezr.. 70 (1976) 167. znd N. N_ U_garova. BiokJtim~wz_ U (1979) 150% R. B. Branchini. T_ M. _Marschner and A. M. Monremurro. Anal. BiocJzem.. 104 (1980) 3S6. S_ T. Thompson, K_ M. Cass and E. Stellwagen. Proc. .Vut. dcd Sci. L-S., 72 (1975) 669. A_ _Antonik, ti_S_ Par_ _-lppl_. 493.874 (1974). S. IM. Chang, K. M. Gooding and F. E. Regnier, J. Chromatogr., 120 (1976) 120. L. D. Ryan and C. S. Vestling, Arch. Biochent. BibpJzys., 170 (1974) 279. A_ Gorg. W. Postel and R. Westermeier, Sci. Tools, 17 (1979) 14. A_ Lundin. A_ Myhrman and G. Linfors, in M. De Luca (Editor). BiofuJninescence Cl!enlil~~lbiesc~~_ Academic Press. New York. 1951. p_ 45% 10 E. Schlulz, M. Ekinga, F. Marx. and R. G. Schirmer. iVature (London), 250 (1974) lZ!O_ II M. J. Adams, A_ McPherson, M. G. Rossman, R. W. Schevitz and A_ J. Wonacott. J_ Mol. Biol.. 51 (1979) 31. 12 %I. Vijayaiakshmi and J_ Porath. J_ Chromatogr., submitted for publication. Y_ N_ Fillipova

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